Species-specific differences exist in human and mouse with respect to X chromosome inactivation (XCI): a developmentally regulated process in all mammals for achieving dosage compensation of X- linked genes. The long non-coding RNA Xist is crucial in establishing XCI as cells of the inner cell mass (ICM) of the pre-implantation blastocyst progress to differentiation. In mouse, Xist is not expressed in the nave pluripotent cells of the ICM: it only becomes induced at the start of differentiation and silences the X chromosome from which it is expressed for the life of the cell. Contrary to this, human XIST is expressed from all X chromosomes of the nave ICM cells without conferring silencing. During differentiation, only one of the two X chromosomes of female human cells retains XIST expression and becomes silenced, similar to mouse. Here we propose to decipher how functions of XIST are changed in early human development. Specifically, we will address how human XIST can induce silencing in differentiated and primed pluripotent (post-XCI) but not in nave pluripotent (pre-XCI) cells, and decipher the molecular mechanisms that enable human XIST to silence the X chromosome. We hypothesize that different RNA species of XIST are expressed in pre- and post-XCI cells due to alternative splicing and/or differential enhancer/promoter usage. We will test this by examining the RNA species expressed in pre- and post- XCI environments using PCR, RNA-seq, RAP-seq, and 5'-RACE. Moreover, the ability of XIST to silence may depend on the presence of certain protein factors differentially expressed in pre- and post- XCI cells. To this end we will perform genome-scale knockout library screen using CRISPR/Cas9 editing to find proteins that, when disrupted, interfere with XIST's ability to initiate silencing. Our work on the X chromosome in the human system will shed light on how fundamental epigenetic processes have changed in evolution.